The quality control (QC) of glycoprotein folding in the endoplasmic reticulum (ER) involves the interplay of a glucosyltransferase (GT) that only glycosylates not properly folded glycoprotein conformers, glucosidase II that removes residues added by GT and two ER resident lectins (calnexin, CNX and calreticulin, CRT) that specifically recognize monoglucosylated glycoproteins. This mechanism prevents exit of not properly folded glycoproteins to the Golgi and enhances glycoprotein folding efficiency and is directly related to the so-called "conformational diseases". To continue our characterization of the structural features that determine the participation of GT in the QC mechanism we now propose to study: (i) the subtle conformational differences, including those that may eventually result in the formation of amyloid fibers, that determine whether glycoproteins are recognized or not in vivo by GT;(ii) the maximum distance between the N-glycan and the structural distortion that allows GT-mediated glucosylation and (iii) whether glycoprotein structural stability determines its fate in the ER, i.e. successfully passing through the QC or being diverted to ER associated degradation (ERAD). For this purpose, we will express several N-glycosylation and stability lysozyme mutants (including some ones leading to the production of amyloids) in S. pombe cells and to follow whether they are recognized by GT, and whether they successfully fold or if, alternatively, they are derived to ERAD. These data, coupled to a structural characterization of the lysozyme mutants will provide information on why cells fail, in certain instances, to derive dangerous, aggregation-prone glycoproteins to degradation and on whether the structural stability of glycoproteins determines their fate in the ER. To continue our characterization of the role of N-glycans on ERAD we will further study the mechanism by which the ER mannosidase, the putative lectins Htm1p/Mnl1p/EDEM and Yos9p and the conformational sensor GT participate in driving irreparably misfolded glycoproteins to degradation. As a model system we chose S. pombe, a yeast that, contrary to what happens in S. cerevisiae, has a QC of glycoprotein folding similar to that occurring in mammalian cells. We plan to further study the proposal, derived from our own results, that the role of ER 1-mannosidase in ERAD is related to a putative lectin capacity of the protein and not to its enzymatic activity and to evaluate the relative importance in both productive glycoprotein folding and in diversion to ERAD of the first, partial glycan deglucosylation-dependent and of the following, reglucosylation, GT-dependent, CNX-glycoprotein interactions.